SF3B1 c.2469G>A, p.(Met823Ile)

Reasoning

PM2 is met at moderate strength under the applicable generic ACMG/AMP 2015 fallback because the variant is absent from the queried population controls in gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.

Rule

Generic ACMG/AMP 2015 fallback: PM2 applies to a variant absent from population controls (or at an extremely low frequency when recessive).

Evidence considered

• The queried normalized alleles (GRCh37 2-198266151-C>T and GRCh38 2-197401427-C>T) were reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.

Still missing

• No SF3B1-specific PM2 specification or disease-specific maximum credible allele frequency was available; the generic ACMG/AMP 2015 PM2 rule was used.

Sources

Reasoning

PP2 applies to missense variants in genes where missense is a common and well-established mechanism of disease and where benign missense variation is not a frequent finding. SF3B1 has a well-documented missense-driven germline disease mechanism: a cohort of 26 individuals with SF3B1-related neurodevelopmental disorder included 17 with heterozygous, largely de novo missense variants (versus 9 with predicted loss-of-function), with missense variants associated with a more severe, syndromic phenotype including cardiac and gastrointestinal anomalies, distinct from the LoF subset and from somatic cancer-associated missense variants. This establishes missense substitution as an established germline pathogenic mechanism for SF3B1, supporting PP2 for this missense variant (p.Met823Ile) in the absence of a gene-specific CSPEC to the contrary.

Rule

generic_ACMG_PP2

Evidence considered

• PMID:41577671 (full-text extract, retrieved via pvs1_gene_context literature search): cohort of 26 individuals with SF3B1 constitutional heterozygous variants; missense variants (n=17) were largely de novo and associated with a more severe/syndromic phenotype than the loss-of-function subset (n=9), establishing missense substitution as a recognized SF3B1 germline disease mechanism distinct from LoF.
• pvs1_gene_context.json mechanism_rationale and supporting_sources confirm SF3B1 germline disease-context literature was reviewed as part of gene-level mechanism assessment.

Still missing

• No formal SF3B1 missense constraint metric (e.g., gnomAD missense Z-score or o/e ratio) was present in the case bundle to quantitatively confirm depletion of benign missense variation.

Sources

Reasoning

PM1 requires the variant to lie in a well-established mutational hotspot or critical, well-studied functional domain without benign variation. p.Met823(Ile) falls within the SF3B1 HEAT-repeat domain broadly, but the case-specific cancerhotspots.org lookup for 'SF3B1 M823' returned no statistically significant hotspot result, and COSMIC returned no somatic recurrence at this residue. No CSPEC/VCEP-defined critical subdomain boundaries for SF3B1 were retrieved (cspec.found=false), and the only literature hotspot data point available (K700E) is a distinct, well-separated codon from M823, not evidence of a hotspot at residue 823 itself.

Rule

generic_ACMG_PM1

Evidence considered

• cancerhotspots.org query 'SF3B1 M823' returned search_status 'no_result', evidence_sentence: 'This variant does not lie in a statistically significant hotspot.'
• COSMIC search for SF3B1 c.2469G>A returned search_status 'no_result', evidence_sentence: 'This variant has not previously been reported in somatic cancers (COSMIC).'
• PMID:41577671 (SF3B1 germline NDD cohort, full-text extract) states somatic cancer SF3B1 missense hotspots recur in the H4-H8 HEAT repeats with K700E being the most frequent hotspot residue; no statement in the retrieved text identifies M823 as part of a recognized hotspot cluster.

Still missing

• No VCEP-specific SF3B1 domain/hotspot boundary framework was available to confirm or refute M823 as a critical functional residue.
• COSMIC and cancerhotspots.org searches for this residue returned no data (searches failed to surface a record, not confirmed truly negative).

Reasoning

No ClinGen VCEP/CSPEC exists for SF3B1 (cspec.found=false; final_classification_framework.source='generic_acmg_fallback'), so generic ACMG/AMP calibrated in-silico rules apply. This is a missense variant, NM_012433.3:c.2469G>A / NP_036565.2:p.(Met823Ile), lying well within the coding exon (selector_short exon c range 2371-2496 spans c.2469) and away from any splice consensus site. REVEL score = 0.571 (local REVEL v1.3 lookup). Per the ClinGen SVI-endorsed REVEL calibration (Pejaver et al. 2022, PMID:36413997), PP3_Supporting requires REVEL >= 0.644; 0.571 falls short and lands in the indeterminate zone between the PP3 and BP4 thresholds, so PP3 is not met on REVEL. SpliceAI predicts no splice-altering effect (max delta score 0.033; DS_AG 0.005, DS_AL 0.033, DS_DG 0.001, DS_DL 0.0), consistent with the variant's deep-exonic location and 'sequence_variant' consequence annotation, but this pipeline has no independently verified, publication-cited SpliceAI threshold for formally invoking PP3, so the SpliceAI result is not used to trigger a splice-path PP3 code -- it is only consistent with absence of splice disruption. BayesDel score (0.150289, local BayesDel_170824_noAF lookup) is available but per pipeline policy has no verified published calibration threshold and is therefore treated as insufficiently calibrated for PP3, regardless of the raw value. No verified aGVGD result is present in this bundle. Overall, no calibrated computational-evidence path (missense or splicing) meets criteria for PP3.

Rule

generic_acmg_pp3_computational

Evidence considered

• REVEL score = 0.571 for NM_012433.3:c.2469G>A (local REVEL v1.3 lookup, source_registry key 'revel'); ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997) sets PP3_Supporting at REVEL >= 0.644, which this score does not reach.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.03)' with DS_AG 0.005, DS_AL 0.033, DS_DG 0.001, DS_DL 0.0 -- no significant splice-altering signal, but no citable calibrated ACMG threshold exists in this pipeline to convert this low score into a formal PP3 trigger.
• BayesDel score 0.150289 (local BayesDel_170824_noAF lookup) noted but excluded from PP3 determination because no verified published calibration threshold is available to this pipeline for BayesDel.

Still missing

• No gene-specific VCEP PP3/BP4 lookup table available for SF3B1 to directly pre-assign a code.
• BayesDel score is present but lacks a verified, citable published calibration threshold usable by this pipeline for PP3.
• No independently verified ACMG/ClinGen SVI-endorsed SpliceAI threshold is available to formally invoke PP3 from the splice score alone; REVEL 0.571 alone does not meet the PP3_Supporting threshold of 0.644 (PMID:36413997).

Sources

Reasoning

ClinVar has no exact-variant record for SF3B1 c.2469G>A (p.Met823Ile); consequently, there is no exact-variant Pathogenic/Likely pathogenic expert-panel assertion to support PP5.

Rule

Apply PP5 only for an exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification.

Evidence considered

• ClinVar exact-variant search returned no result for NM_012433.3:c.2469G>A.

Sources

Reasoning

BA1 is not met: the variant is absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0, so no population allele frequency reaches a stand-alone benign threshold.

Rule

Generic ACMG/AMP 2015 fallback: BA1 requires an allele frequency above the stand-alone benign threshold.

Evidence considered

• The queried normalized alleles (GRCh37 2-198266151-C>T and GRCh38 2-197401427-C>T) were reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0.

Still missing

• No SF3B1-specific BA1 threshold or disease-specific maximum credible allele frequency was available; this does not affect the conclusion because the variant is absent in all queried population datasets.

Sources

Reasoning

BS1 is not met: the variant is absent from all queried population datasets and therefore cannot be more frequent than expected for an SF3B1-associated disorder.

Rule

Generic ACMG/AMP 2015 fallback: BS1 requires an allele frequency greater than expected for the disorder.

Evidence considered

• The variant was reported absent from gnomAD v2.1, gnomAD v4.1, and gnomAD-Canada v1.0; no overall or ancestry-specific allele frequency is available to exceed a benign strong threshold.

Still missing

• No SF3B1-specific BS1 threshold, disease prevalence, inheritance model, or penetrance estimate was supplied; these are needed to define a quantitative gene-specific BS1 cutoff but cannot convert absence into BS1 evidence.

Sources

Reasoning

BP1 applies when a missense variant occurs in a gene for which primarily truncating/loss-of-function variants are known to cause disease (i.e., missense is not an established mechanism). This is not the case for SF3B1: the germline neurodevelopmental disorder literature documents missense variants as a common and even more severe-phenotype-associated mechanism (17/26 patients) alongside the LoF subset (9/26), and SF3B1 is additionally a well-known somatic missense cancer driver gene (recurrent hotspot substitutions such as K700E). Because missense is an established, non-incidental disease mechanism for SF3B1, BP1 does not apply.

Rule

generic_ACMG_BP1

Evidence considered

• PMID:41577671 (full-text extract): 17/26 SF3B1 germline NDD cohort patients carried missense variants, associated with a more severe/syndromic phenotype than the LoF subset (9/26), demonstrating missense is an established, non-rare SF3B1 disease mechanism, disqualifying BP1 applicability.

Sources

Reasoning

No ClinGen VCEP/CSPEC exists for SF3B1 (cspec.found=false; framework 'generic_acmg_fallback'), so generic ACMG/AMP calibrated in-silico rules apply. REVEL score = 0.571 (local REVEL v1.3 lookup). Per the ClinGen SVI-endorsed REVEL calibration (Pejaver et al. 2022, PMID:36413997), BP4_Supporting requires REVEL <= 0.290; 0.571 is well above this threshold and instead sits in the indeterminate zone between the PP3 and BP4 cutoffs, so BP4 is not met on REVEL. SpliceAI shows no splice-altering signal (max delta score 0.033), which is directionally consistent with no functional impact via splicing, but this pipeline has no independently verified, publication-cited SpliceAI threshold that would let this observation formally satisfy BP4 on its own, so it is treated as supportive context only, not a standalone trigger. BayesDel score (0.150289) is available but per pipeline policy has no verified published calibration threshold, so it is treated as insufficiently calibrated for BP4 regardless of the raw value, exactly as for PP3. No calibrated computational-evidence path (missense or splicing) supports BP4 for this variant.

Rule

generic_acmg_bp4_computational

Evidence considered

• REVEL score = 0.571 for NM_012433.3:c.2469G>A (local REVEL v1.3 lookup, source_registry key 'revel'); ClinGen SVI calibration (Pejaver et al. 2022, PMID:36413997) sets BP4_Supporting at REVEL <= 0.290, which this score exceeds.
• SpliceAI evidence_sentence: 'SpliceAI predicts no significant splice impact for this variant (max delta score = 0.03)' -- consistent with no splicing effect but not used to independently trigger BP4 absent a citable calibrated ACMG SpliceAI threshold.
• BayesDel score 0.150289 (local BayesDel_170824_noAF lookup) noted but excluded from BP4 determination because no verified published calibration threshold is available to this pipeline for BayesDel.

Still missing

• No gene-specific VCEP PP3/BP4 lookup table available for SF3B1 to directly pre-assign a code.
• BayesDel score is present but lacks a verified, citable published calibration threshold usable by this pipeline for BP4.
• REVEL 0.571 exceeds the BP4_Supporting threshold of <=0.290 (PMID:36413997), placing the variant in an indeterminate zone for both PP3 and BP4.

Sources

Reasoning

ClinVar has no exact-variant record for SF3B1 c.2469G>A (p.Met823Ile); consequently, there is no exact-variant Benign/Likely benign expert-panel assertion to support BP6.

Rule

Apply BP6 only for an exact-variant ClinVar expert-panel Benign or Likely benign classification.

Evidence considered

• ClinVar exact-variant search returned no result for NM_012433.3:c.2469G>A.

Sources

Reasoning

PS1 requires a different nucleotide change producing the identical amino acid change (p.Met823Ile) already established as pathogenic. This variant (NM_012433.3:c.2469G>A) is absent from ClinVar (no_result search status), so no comparator pathogenic classification exists for the same amino-acid change. No other source in this case bundle documents an established-pathogenic p.Met823Ile allele.

Rule

generic_ACMG_PS1

Evidence considered

• ClinVar search for SF3B1 c.2469G>A returned search_status 'no_result' / found: false, evidence_sentence 'This variant is absent from ClinVar.'

Still missing

• No ClinVar record for this variant or any other nucleotide change producing p.Met823Ile.
• No literature-confirmed pathogenic variant with the identical amino-acid change was identified in the case bundle.

Sources

Reasoning

No case-level proband phenotype, parental genotype, parentage confirmation, or mosaicism testing data were provided. Confirmed de novo occurrence and phenotype consistency therefore cannot be assessed.

Rule

Generic ACMG/AMP fallback: PS2 requires a de novo variant in an affected individual with phenotype consistent with the gene/disease; the provided bundle lacks the required case evidence.

Evidence considered

• The consolidated case-evidence bundle reports no SF3B1 CSPEC/VCEP framework and contains no case-level trio, parental-testing, phenotype, or mosaicism evidence for NM_012433.3:c.2469G>A.

Still missing

• Proband phenotype and diagnostic indication.
• Genotypes of both biological parents using an appropriate assay.
• Confirmation of biological parentage.
• Assessment of possible parental or proband mosaicism, when clinically indicated.

Reasoning

No validated functional assay data (splicing assay, minigene, complementation assay, or other well-established functional study) for NM_012433.3:c.2469G>A (p.Met823Ile) was found in the case evidence bundle. No CSPEC/VCEP functional framework is available for SF3B1 (framework_mode: generic_acmg; cspec.found: false), so no gene-specific PS3 assay calibration exists to apply. Literature search returned 0 PMIDs in the literature_packet, and the PVS1 gene-context literature papers retrieved (germline SF3B1 disease-association searches) are gene-level only and do not mention this specific variant or report functional assay results for it.

Still missing

• No functional assay (in vitro or in vivo) data available for this specific variant
• No SF3B1-specific VCEP/CSPEC functional evidence framework available to calibrate assay strength
• No literature identified that experimentally characterizes p.Met823Ile

Reasoning

No case-control or other statistically evaluable enrichment data for SF3B1 c.2469G>A (p.Met823Ile) were identified in the supplied evidence bundle.

Rule

Generic ACMG/AMP PS4: prevalence in affected individuals must be significantly increased compared with controls.

Evidence considered

• No applicable SF3B1 VCEP/ClinGen gene-specific specification was supplied; generic ACMG/AMP criteria were used.
• The literature pass contains no PMIDs or variant-specific publications, and no case-control dataset is provided.

Still missing

• Variant-specific affected-case counts and ancestry-matched control counts, with a valid enrichment analysis, are required.

Reasoning

PM3 cannot be assessed: the case bundle contains no affected-proband observations of NM_012433.3:c.2469G>A with a pathogenic/likely pathogenic SF3B1 variant in trans, no phase determination, and no established recessive disease context applicable to this observation.

Rule

Generic ACMG/AMP PM3: requires an observation in trans with a pathogenic variant in an affected individual with a recessive disorder.

Evidence considered

• No SF3B1 ClinGen CSPEC, VCEP material, or local gene-specific framework was retrieved; the bundle specifies generic ACMG/AMP fallback.
• The literature packet contains no variant-specific PMIDs, and ClinVar has no record for NM_012433.3:c.2469G>A.
• No source in the bundle documents an affected individual carrying this variant with a pathogenic or likely pathogenic SF3B1 variant in trans.

Still missing

• Affected-proband genotype data, including any second SF3B1 variant.
• Parental or read-based phase establishing whether the variants are in trans.
• Disease-specific inheritance and phenotype evidence supporting a recessive SF3B1 condition for the proband.

Sources

Reasoning

PM5 requires a different missense change at the same residue (p.Met823X) previously established as pathogenic. The case's pm5_candidates artifact explicitly flags that classic same-residue PM5 semantics could not be safely confirmed for this case ('eligible_for_classic_pm5_search': false) and instructs reviewers not to apply PM5 from that artifact alone, with zero same-residue candidates harvested (same_residue_candidates: 0).

Rule

generic_ACMG_PM5

Evidence considered

• pm5_candidates_summary: pm5_mode 'unknown', eligible_for_classic_pm5_search=false, recommendation level 'not_applicable', summary: 'Unable to confirm classic same-residue PM5 semantics safely; skipping automatic candidate harvesting.' candidate_summary shows total_candidates_seen=0, same_residue_candidates=0.

Still missing

• PM5 comparator harvesting was not completed/confirmed for this case; no same-residue alternate-amino-acid pathogenic variants were identified.
• No ClinVar or literature search specific to alternate M823 substitutions was independently performed beyond the flagged pm5_candidates artifact.

Sources

Reasoning

No report of de novo occurrence, parental testing, or parentage status was provided. Assumed de novo occurrence cannot be established.

Rule

Generic ACMG/AMP fallback: PM6 requires an assumed de novo occurrence when parental confirmation is unavailable; no such occurrence is documented in the provided evidence.

Evidence considered

• The consolidated case-evidence bundle contains no parental genotype data or case report describing de novo occurrence of NM_012433.3:c.2469G>A.

Still missing

• Report of testing in both biological parents demonstrating absence of the variant, if available.
• Whether parentage was confirmed; if not, documentation sufficient to support an assumed de novo assessment.
• Proband phenotype and its consistency with an SF3B1-associated condition.

Reasoning

No informative relatives, genotypes, phenotypes, or meioses were provided. Familial co-segregation cannot be evaluated.

Rule

Generic ACMG/AMP fallback: PP1 requires co-segregation with disease in multiple affected family members; no segregation data are available.

Evidence considered

• The consolidated case-evidence bundle contains no pedigree, relative-level genotype, or segregation data for NM_012433.3:c.2469G>A.

Still missing

• Pedigree with affected and unaffected relatives.
• Variant genotypes for informative relatives.
• Phenotype status and age at evaluation for tested relatives.
• Number of informative meioses and any phenocopies.

Reasoning

The supplied record contains no phenotype for an individual carrying SF3B1 c.2469G>A (p.Met823Ile), and no variant-specific phenotype series. Therefore phenotype specificity cannot be assessed.

Rule

Generic ACMG/AMP PP4: the patient's phenotype or family history must be highly specific for a disease with a single genetic etiology.

Evidence considered

• No applicable SF3B1 VCEP/ClinGen gene-specific specification was supplied; generic ACMG/AMP criteria were used.
• The literature pass contains no PMIDs or variant-specific publications.

Still missing

• A documented proband phenotype and disease-specificity assessment for the relevant SF3B1 germline disorder are required.

Reasoning

BS2 is not assessed because no observations of this variant in well-phenotyped unaffected individuals, including age and disease-relevant phenotype information, were provided.

Rule

Generic ACMG/AMP 2015 fallback: BS2 requires observation in a healthy adult individual for a fully penetrant early-onset disorder.

Evidence considered

• Population-database absence provides no unaffected-carrier or homozygote observations and cannot establish BS2.

Still missing

• Variant-level observations in adequately aged, well-phenotyped unaffected individuals are required.
• SF3B1 disease-specific penetrance, age of onset, and phenotype definition are needed to judge whether an unaffected observation would be informative.

Reasoning

No validated functional assay data showing normal/wild-type function for NM_012433.3:c.2469G>A (p.Met823Ile) was found in the case evidence bundle. No CSPEC/VCEP functional framework is available for SF3B1 (framework_mode: generic_acmg; cspec.found: false). Literature search returned 0 PMIDs in the literature_packet, and no retrieved publication reports functional characterization of this specific variant.

Still missing

• No functional assay data available for this specific variant showing normal function
• No SF3B1-specific VCEP/CSPEC functional evidence framework available to calibrate assay strength
• No literature identified that experimentally characterizes p.Met823Ile

Reasoning

No unaffected variant-positive relatives, affected variant-negative relatives, or other informative non-segregation observations were provided. Lack of segregation cannot be evaluated.

Rule

Generic ACMG/AMP fallback: BS4 requires convincing lack of segregation with disease; no informative family data are available.

Evidence considered

• The consolidated case-evidence bundle contains no family genotype-phenotype observations that demonstrate or refute segregation of NM_012433.3:c.2469G>A.

Still missing

• Pedigree and genotype-phenotype data from informative relatives.
• Age-dependent penetrance and phenocopy assessment before interpreting any apparent non-segregation.
• Confirmation that the tested familial phenotype is attributable to the relevant SF3B1-associated condition.

Reasoning

BP2 cannot be assessed: the case bundle contains no co-occurring pathogenic/likely pathogenic variant, no cis/trans phase evidence, and no unaffected or phenotype-discordant carrier observation relevant to SF3B1 disease.

Rule

Generic ACMG/AMP BP2: requires documented co-occurrence with a pathogenic variant in a configuration and disease context that supports a benign interpretation.

Evidence considered

• No SF3B1 ClinGen CSPEC, VCEP material, or local gene-specific framework was retrieved; the bundle specifies generic ACMG/AMP fallback.
• The literature packet contains no variant-specific PMIDs, and ClinVar has no record for NM_012433.3:c.2469G>A.
• No source in the bundle reports this variant in cis with a pathogenic or likely pathogenic variant, in trans with a pathogenic variant for a dominant disorder, or in an unaffected individual.

Still missing

• Complete proband genotype data for pathogenic or likely pathogenic variants that co-occur with NM_012433.3:c.2469G>A.
• Parental or read-based phasing to establish cis or trans configuration.
• Phenotype, segregation, and inheritance-mode data to evaluate whether any co-occurrence is non-causal.

Sources

Reasoning

No case-level molecular diagnosis or phenotype attribution is provided. An alternate molecular cause explaining a relevant phenotype cannot be evaluated.

Rule

Generic ACMG/AMP BP5: a variant is found in a case with an alternate molecular basis for disease.

Evidence considered

• No applicable SF3B1 VCEP/ClinGen gene-specific specification was supplied; generic ACMG/AMP criteria were used.

Still missing

• Case-level phenotype information and testing results establishing an alternative molecular diagnosis are required.